Scrapie and related transmissible spongiform encephalopathies result in the accumulation of an abnormal protease-resistant form of an endogenous brain protein called PrP. This protease-resistant PrP (PrP-res) can aggregate into fibrils and form amyloid-like plaques. There is substantial evidence that the formation of PrP-res is scrapie-specific and important in the pathogenesis of the disease. However, it is not yet clear whether Prp-res is the transmissible agent itself, a component of the agent, or a byproduct of the infection which happens to co-fractionate with scrapie infectivity. Since the replication or pathogenesis of the scrapie agent may involve the production of PrP-res, we have continued studies of the properties and biosynthesis of both PrP-res and the normal, protease-sensitive form of PrP in scrapie-infected neuroblastoma cells. In the past year, we have determined that PrP-res is synthesized much more slowly than the normal Prp isoform, and thus may be generated from it. This was supported by experiments showing that PrP-res is made from a precursor that, like the normal isoform, resides on the cell surface in a phospholipase-and protease-sensitive state. Once it is made, PrP-res is truncated at the N- terminus by lysosomal proteases, and therefore must be translocated to the lysosomes. When considered together, these findings imply that the conversion of PrP to the protease-resistant state is a post-translational event that occurs at the cell surface or along the endocytic pathway to the lysosomes. The molecular nature of the conversion of PrP to a protease-resistant form remains a mystery. It is possible that a conformational abnormality accounts for the characteristics of PrP-res. To investigate this possibility, we have used a recently developed Fourier transform infrared spectroscopy technique to analyze the secondary structure of the proteinase K-resistant core of PrP-res as it exists in highly infectious fibril preparations. The analysis indicated that PrP-res is composed primarily of beta-sheet (47%), which is consistent with its amyloid-like properties. In addition, significant amounts of turn and alpha-helix were identified, indicating that amyloid fibrils need not be exclusively beta-sheet. The infrared-based secondary structure compositions were then used as constraints to improve the theoretical localization of the secondary structures within the PrP-res molecule. We are now developing a method of purifying the normal PrP isoform under nondenaturing conditions so that we will be able to compare its conformation to that of PrP-res.